Organisms encode multiple Hsp70s to increase capacity to regulate abundance of Hsp70 in accordance with need and to provide a range of distinct Hsp70 functions for carrying out specific tasks within all cells or in distinct cell types. We constructed a yeast system to evaluate Hsp70s from any source and are using it to investigate how Hsp70s within and across species influence propagation of amyloid in vivo and act in cellular protein quality control (PQC). The wide range of responses that prions have to alterations of Hsp70s and their co-chaperones provides a sensitive way to investigate even subtle functional distinctions among highly redundant Hsp70s and an approach to uncover the underlying mechanisms. Hsp90 is another major and essential PQC chaperone that has essential roles in many fundamental cellular processes. Many of its functions rely on interaction and cooperation with the Hsp70 system. Some Hsp90 client substrates bind first to Hsp70, which transfers them to Hsp90 for continued processing. We earlier showed the Hsp70/Hsp90 co-chaperone Sti1 (Hop in mammals) can regulate Hsp70 and Hsp90 independently and that it must bind both Hsp70 and Hsp90 in order for processes that depend this client transfer to function efficiently. By developing mass spectrometry methods and using genetics, biochemistry, molecular biology to analyze physical and functional interactions in vivo and in vitro we found that Sti1 had two distinct roles in regulating function of the Hsp90 reaction cycle. Our findings clarify the roles of Sti1 as an Hsp70-Hsp90 co-chaperone and provide important insight into mechanistic details of how Hsp90 progresses in its reaction cycle. In collaboration with another group at NIH we also showed that physical interaction of yeast Hsp70, even without Sti1, is important for Hsp90 functions in vivtro and in vivo. We also are working to determine if differences in functions of Hsp70s are mediated by ways they cooperate with J-proteins, other co-chaperones, such as Hsp70 nucleotide exchange factors, or other major chaperones, such as Hsp90. We are working to learn whether such differences in Hsp70 function contribute to protection from amyloid toxicity that we see in some of our strains and if human chaperones possess such protective functions. Altering abundance or function of Hsp70 or Hsp90 can moderate pathology in models of protein folding disorders, while in the same models reducing chaperone activity can exacerbate, or alone even cause pathology. Hsp70 and Hsp90 therefore are promising therapeutic candidates for amyloid and other protein folding disorders and they are being evaluated extensively as drug targets. Altering Hsp70 and Hsp90 co-chaperones also moderates pathology in several models of amyloid and other protein folding disorders. Our findings can help guide decisions about which Hsp70/90-family members would be most useful for such applications, or identify potential problems that could arise due to distinct sensitivities of different Hsp70s or Hsp90s to specific compounds. Overall our work provides insight into functions of these chaperones that can help guide strategies for using chaperones as targets for therapy in such diseases.